Benchmarking of SpCas9 variants enables deeper base editor screens of BRCA1 and BCL2

Annabel K. Sangree, Audrey L. Griffith, Zsofia M. Szegletes, Priyanka Roy, Peter C. DeWeirdt, Mudra Hegde, Abby V. McGee, Ruth E. Hanna and John G. Doench ()
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Annabel K. Sangree: Broad Institute of MIT and Harvard
Audrey L. Griffith: Broad Institute of MIT and Harvard
Zsofia M. Szegletes: Broad Institute of MIT and Harvard
Priyanka Roy: Broad Institute of MIT and Harvard
Peter C. DeWeirdt: Broad Institute of MIT and Harvard
Mudra Hegde: Broad Institute of MIT and Harvard
Abby V. McGee: Broad Institute of MIT and Harvard
Ruth E. Hanna: Broad Institute of MIT and Harvard
John G. Doench: Broad Institute of MIT and Harvard

Nature Communications, 2022, vol. 13, issue 1, 1-17

Abstract: Abstract Numerous rationally-designed and directed-evolution variants of SpCas9 have been reported to expand the utility of CRISPR technology. Here, we assess the activity and specificity of WT-Cas9 and 10 SpCas9 variants by benchmarking their PAM preferences, on-target activity, and off-target susceptibility in cell culture assays with thousands of guides targeting endogenous genes. To enhance the coverage and thus utility of base editing screens, we demonstrate that the SpCas9-NG and SpG variants are compatible with both A > G and C > T base editors, more than tripling the number of guides and assayable residues. We demonstrate the performance of these technologies by screening for loss-of-function mutations in BRCA1 and Venetoclax-resistant mutations in BCL2, identifying both known and new mutations that alter function. We anticipate that the tools and methodologies described here will facilitate the investigation of genetic variants at a finer and deeper resolution for any locus of interest.

Date: 2022
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DOI: 10.1038/s41467-022-28884-7

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